Summary Regulation of genome expression is essential for cells to maintain their identity and loss of cell identity leads to tumorigenesis. Cancer Genome Project has revealed that chromatin modifiers and remodelers are highly mutated in human cancers, however, despite the importance of these factors, we know little about their mechanisms of activity. To address this knowledge gap, we will combine cryo-EM, biochemistry and genetics to determine how enzymes and structural proteins modify nucleosome and chromatin structure. Among the key players in the control of genome expression are histone modifications that, through specific reader domains, recruit various protein complexes to chromatin. Histone lysine methylation is a stable chromatin mark that is deposited by histone lysine methyltransferases (KMTs) and methylation of different lysine residues has different outcomes on gene expression; H3K36 methylation is deposited over actively transcribed genes, whereas H3K9 methylation is a hallmark of silent heterochromatin. Perturbations in KMT levels lead to aberrant genome expression and formation of cancers cells, however, we do not understand how KMTs bind and modify nucleosome. H3K9 methylation and heterochromatin are required for deposition of centromere specific histone H3 variant CENP-A to chromatin, an epigenetic mark of centromeres. The centromere is the specialized chromatin region on which kinetochores assemble to segregate chromosomes. Despite the importance, structure of centromeric chromatin assembled on CENP-A nucleosome is still largely unknown. Guided by the strong preliminary data, we propose to pursue three Specific Aims to understand how KMTs bind and modify nucleosomes and to characterize centromeric chromatin. We will combine cryo-EM with biochemistry and genetics to determine mechanisms of H3K36 (Aim 1) and H3K9 (Aim 2) methylation. Moreover, we will use cryo-EM to visualize centromeric chromatin assembled on CENP-A nucleosome (Aim 3). Together, our proposed studies will have broad impact in chromatin field by showing how chromatin proteins bind the nucleosome and how this interaction provides specificity for their activity. Our long-term goals are to understand the regulation of genome expression by chromatin and discover why mutations in chromatin proteins lead to the formation of cancer cells.